Methods, agents and kits for detection of organophosphorus compounds

ABSTRACT

Novel generic antibodies are provided that are specific to groups of organophosphates, particularly to a class of organophosphate pesticides in common usage. Further provided are compounds and methods for raising these antibodies, test kits containing them and methods for their use. The antibodies targeted at low molecular weight (i.e. below MW 1000) organophosphorous pesticides methacrifos, pirimphos methyl, etrimfos, fenitrothion, chlopyrifos methyl, dichlorovos and malathion. Preferred conjugates of the invention are of the formula (RO 2  --P(S)--Z--[Y]--Protein wherein R is lower alkyl and Z is as O, S or --NH--, Y is a spacer group and &#34;Protein&#34; indicates a protein suitable for use in hapten protein conjugates for the purpose of raising antibodies and antisera. Typical and preferred proteins are bovine serum albumin, ovalbumin from chicken egg, keyhole limpet hemocyananin and mixtures of these.

This is a division of application Ser. No. 08/557,003, filed Apr. 26,1996, now U.S. Pat. No. 5,830,770 issued Nov. 3, 1998 which is a 371 ofPCT/GB94/01060 filed May 18, 1994.

The present invention relates to novel generic antibodies that arespecific to groups of organophosphates, particularly to a class oforganophosphate pesticides in common usage. Further provided are haptencompounds and methods for using them in raising these antibodies, testkits containing them and methods for their use.

A number of immunoassays are available for the detection of variousbiologically important compounds. Such compounds include the importantorganophosphate (OP) class of pesticides (see eg. WO 91/00294). Theseimmunoassays are relatively sensitive, but are directed againstindividual OP molecules and thus several separate assays are required toeffectively screen a material for OP presence.

A group of these organophosphate pesticides that is commonly used forfoodstuff, eg. grain, protection comprises the compounds methacrifos,pirimphos methyl, fenitrothion, etrimfos, propetamphos, chlopyrifosmethyl and dichlorvos, and screening a material for the presence of anyof these potentially toxic compounds conventionally requires use ofseven separate agents. While these compounds share some chemicalfeatures with each other, including that of the OP group itself, othercommonly used pesticides are distinguished by variant OP groups. Inmalathion for example a (CH₃ O)₂ P(S)--S-- group is present as comparedto the (CH₃ O)₂ P(S)--O-- group of the compounds referred to above.

The present inventors have now provided a novel strategy for raisingantibodies such that they are specifically directed to bind with two ormore of these specified pesticidal organophosphorous compounds andfurther provide methods for using these and kits containing them, suchthat materials can be screened for presence of a member of a selectedgroup with a single antibody reagent. Still further they have providedorganic haptens and their conjugates which may be used specifically toraise these antibodies, or antisera containing them. as eithermonoclonal or polyclonal antibodies. Particularly provided areantibodies capable of specific binding with two or more, more preferablythree or more, and most preferably all of the compounds methacrifos,fenitrothion, etrimfos, propetamphos, dichlorvos and optionally one ormore of malathion, dimethoate, chlorfenvinphos, chlopyrifos methyl,tetrachlorvinphos and pirimiphos-methyl, wherein the relative bindingaffinity with each of these bound compounds is such that a singlestrength antibody reagent can be used to bind them. Preferably the OPsbound are capable of displacing percentages of bound hapten from theantibody at a concentration of 100 μg/ml within a factor of 20, morepreferably of 5, and more preferably 2 of each other. Also provided arehaptens and hapten-protein conjugates which evoke production of suchantibodies when adminstered to animals.

The concept that one generic antibody or antisera might be raised to becapable of detecting a number of organophosphates has been considered onone occasion previously by Suedi and Heesham Kiel: (1988) MilchwirtForsch 40 179-202, but this involved use of a number of model pesticidemolecules to provide a number of protein conjugates. Furthermore theantibody reagents obtained were not capable of use in reliablescreening.

In a first aspect of the present invention there is provided a haptenprotein conjugate capable of stimulating production of antibodies whichhave specific binding affinity for compounds including the chemicalmoiety I:

    (RO).sub.2 P(S)--A--                                       (I)

wherein R is lower alkyl, particularly C₁₋₄ alkyl, and A is O or S;characterised in that the antibody binds two or more, preferably threeor more, and most preferably all of the compounds methacrifos,fenitrothion, propetamphos, dichlorvos, dimethoate, malathion,chlorfenvinphos and etrimfos; preferably also binding to chlopyrifosmethyl, tetrachlorvinphos and pirimiphos-methyl.

Most preferably the conjugates evoke production of antibodies targetedat moiety (I) where R is methyl, and preferably where A is O, whereincross-reactivity with other compounds relative to those having thismoiety is low enough to enable ready identification of the targetedcompounds in a mixture by binding with a known antibody concentration.

Preferred hapten protein conjugates of the invention are of formula(II):

    (RO).sub.2 --P(S)--Z--[Y]--Protein (II)

wherein R is as defined for moiety (I) and Z is O, S or --NH--, Y is aspacer group and `Protein` indicates a protein suitable for use inhapten-protein conjugates for the purpose of raising antibodies andantisera. Typical and preferred proteins are bovine serum albumin,ovalbumin from chicken egg, keyhole limpet hemocyanin and mixtures ofthese. Preferably linkage to protein is by one of its amino groups.Preferred spacer groups are format --(CH₂)_(n) --B-- wherein n is suchthat the carbon chain length is 4 to 6 and B is CO or O(CO). or thespacer is a pyridine or pyrimidine ring with a CO or O(CO) group.Optionally the spacer may have side chains, eg. as in compounds such asβ-alanine as disclosed by WO 9100294, but the most preferred conjugatesof the invention have a straight carbon chain of 4-6 eg. a1,4-butanediol spacer whereby Z is O and B is --O(CO)-- provided by wayof carbodiimide activation of the --OH end group. Examples of suitablehaptens are listed as follows:

    ______________________________________                                        (CH.sub.3 O).sub.2 --P(S)--O--(CH.sub.2).sub.4 --O--CO--NH-Protein                                       (butandiol                                            spacer)                                                                      (CH.sub.3 O).sub.2 --P(S)--O--(CH.sub.2).sub.4 --CO--NH-Protein                                        (butandiol                                            spacer)                                                                      (CH.sub.3 O).sub.2 --P(S)--O--CH.sub.2 --CH.sub.2 --O--CO--NH--Protein                                 (3 carbon                                             spacer)                                                                      (CH.sub.3 O).sub.2 --P(S)--O--CH.sub.2 --CH.sub.2 --CO--NH-Protein (3                                  carbon                                                spacer)                                                                      (CH.sub.3 O).sub.2 --P(S)--O--CH.sub.2 --O--CO--NH-Protein (2 carbon                                     spacer)                                            (CH.sub.3 O).sub.2 --P(S)--O--CH.sub.2 --CO--NH-Protein (2 carbon                                        spacer)                                            (CH.sub.3 O).sub.2 --P(S)--S--CH.sub.2 --CH.sub.2 --O--CO--NH-Protein                                  (SH-linked                                            spacer)                                                                      (CH.sub.3 O).sub.2 --P(S)--S--CH.sub.2 --CH.sub.2 --CO--NH-Protein                                     (SH-linked                                            spacer)                                                                      (CH.sub.3 O).sub.2 --P(S)--O-pyridine-O--CO--NH-Protein (pyridine                                        spacer)                                            (CH.sub.3 O).sub.2 --P(S)--O-pyridine-CO--NH-Protein (pyridine                 spacer)                                                                      (CH.sub.3 O).sub.2 --P(S)--O-pyrimidine-O--CO--NH-Protein (pryrimidine                                   spacer)                                            (CH.sub.3 O).sub.2 --P(S)--O-pyrimidine-CO--NH-Protein (pryrimidine                                      spacer)                                          ______________________________________                                    

In a second aspect of the invention there are provided hapten compoundsof formula (III) for use in preparing compounds (II):

    (RO).sub.2 --P(S)--Z--(Y)--B--(D)                          (III)

wherein R, Z, B and Y are as described above and D is H or an activatorgroup. Typical activator groups are those such as imidazole residues ofthe type left by activation with carbodiimides, but halogen atoms mayalso used where B is CO, such as to form an acid halide, capable ofactivating reaction between the compound and protein amino groups.Particularly provided are hapten compounds of formula (IV):

    (RO).sub.2 P(S)--O--(Y)--B--(D)                            (IV)

wherein Y is a spacer of four carbon length, with or without sidechains, preferably being a --(CH₂)₄ -- moiety. Most preferably B isO--CO-- and D is an imidazole residue. Where D is H the compound is anunactivated hapten, otherwise the compound is in the activated form.

Further provided by the present invention is a method for synthesis ofthe haptens and conjugates of formula (II), (III) and (IV) wherein adialkyl halothiophosphate is reacted with a spacer group precursor toproduce the hapten. The resultant hapten is then activated with anactivator moiety to provide the activated product and that is conjugatedwith the protein to provide a protein conjugate of formula (II) in theusual manner.

The dialkyl halothiophosphate/spacer group reaction is convenientlycarried out in organic solvent phase with a base, eg. pyridine, and atlow temperature, eg between -5 and 10° C., preferably 0 and 5° C.Acetone solvent is conveniently used. The reaction mixture is preferablyshaken or stirred with the spacer group being dissolved in thesolvent/base mixture and the dialkyl halothiophosphate being addedslowly to this before reflux, that preferably being at about 60° C. fora period of several, but most preferably about two, hours. Theactivation of the hapten is conveniently carried out using acarbodiimide, eg. carbonyl diimidazole, in the known manner and thisactivated hapten is then conjugated with the selected protein.

The antibodies and antisera produced using these conjugates of theinvention can be raised by any of the methods conventionally used in theart using any of the conventional animal donors. Furthermore theantibodies so raised may be polyclonal or monoclonal; the latter beingderivable by hybridoma technology as will be well understood by thoseskilled in the art. For OP formulae see BRIEF DESCRIPTION OF THEDRAWINGS FIGS. 1A, 1B, 1C, 2A, 2B, 2C, 2D, and 2E.

The present invention further provides test kits comprising theantibodies or antisera of the invention together with other itemsspecific for the determination of the presence of the targetorganophosphates in a material. Such items may include calibrationreagents containing a known amount of target OP compound, or may takethe form of antibodies immobilised on a substrate, eg. on microtitrewell plates or the like, whereby absorbance values may be readilyobtained at suitable wavelength, eg. 450nm, to determine antibody titre,or other reagents such as specific buffers, blocking agents or colourdevelopers. Typical kit formats are as discussed in W09100294.

The production of antibodies and the antisera containing them, and theiruse in assays, is carried out using known methods and is exemplified inthe Examples. The haptens, conjugates, antibodies, antisera and methodsof the present invention will now be described by way of example only byreference to the following non-limiting Examples.

EXAMPLE 1

Synthesis of generic organophosphate antibody generating conjugatehapten intermediate of formula (III) and (IV).

10 mmoles of pyridine (0.79 g) were dissolved in 5 ml acetone and thesolution kept on ice. To this was added 10 mmoles of 1,4 butanediol(0.90 g) and with constant shaking of the chilled mixture using amagnetic stirrer 10 mmoles of dimethylchlorothiophosphate (CH₃ O)₂--P(S)Cl (1.61 g) was slowly added. The reactants were refluxed at 60°C. for 2 hours and then left overnight at room temperature (Reaction A).

The final product was purified by extracting the mixture of reactantswith 3×25 ml of diethylether, washing that with 3×10 ml of H₂ O and thenevaporating it to a final volume of about 2 to 3 ml. Formation of (CH₃O)₂ P(S)--O--(CH₂)₄ OH (mol wt. 214) was indicated on TLC (Rf=0.l inchloroform:acetone 70:30) and confirmed on GC/MS. ##STR1##

EXAMPLE 2

Activation of hapten.

The ether product solution from Example 1 was treated with 10 mmoles(1.62 g) of carbonyl diimidazole thus providing activated haptensolution. (NB. Where B is a carboxyl group thionyl halide may be used toproduce the corresponding acid halide in the known manner).

EXAMPLE 3

Formation of protein conjugates

3 mmoles of activated hapten from Example 2, in the form of 1 ml of theethereal solution, was added very slowly--one drop every fiveminutes--to each of three protein solutions whilst constantly stirringusing a magnetic stirrer; these containing 30 μmoles of BSA orOvalbumin, or 3 nmoles of Keyhole Limpet hemocyanin respectively in 10ml phosphate buffer saline pH 7.4. Reactants were stirred slowly for 2hours, left overnight at room temperature and then at 4° C. for 72 hours(see Reaction B). The conjugate solutions were analysed for theconcentration of protein using the Coomassie blue method of Bradford:Analytical Biochem. (1976) 72:248-254. The ratio of the conjugation wasfound by the free amino group method of Habeeb as in AnalyticalBiochem(1966) 14:328-336 as an OP:BSA of 10 and an OP:Ovalbumin of 4.

Purification of conjugate was effected by application to Sephadex (PD10) columns, with dialysis of insoluble or sparingly soluble conjugatesagainst distilled water being carried out as a preparatory step prior toapplication. ##STR2##

EXAMPLE 4

Production of polyclonal antibodies to generic conjugate.

Simone Noir half lop rabbits were housed individually and allowed 3weeks to settle in before pre-immunisation blood samples were taken andthe immunisation schedule started. Hapten-Bovine serum albumin (BSA)conjugates were first mixed with equal volumes of Freund's adjuvant sothat the final concentration was about 1 mg/ml of protein.

After testing to show the absence of native antibodies reactive with theconjugates, each rabbit was given subcutaneous injections of 1 ml of theBSA generic conjugate of Example 3 spread over 4 sites (0.25 ml persite) in complete adjuvant. Subsequent injections were given at 4, 8 and12 weeks in incomplete adjuvant. When the antibody titre started to fallthe rabbits were boosted with a further 1 ml of conjugate in Freund'sincomplete adjuvant spread over 4 sites. In the tables below rabbitsR19,20,21 were immunised with this generic conjugate, ratio OPhapten:protein of 10:1, of the invention. Blood was taken from themarginal ear vein prior to immunisation for antibody checks and at 4 and14 weeks after injection in order that antisera stocks could be builtup. Antisera were prepared by allowing blood to clot overnight at 4° C.and then centrifuging it to provide clear fluid. Antibodies could beisolated from this as required by use of ion exchange or affinity columnchromatography with immobilised target organophosphate groups in theconventional manner.

EXAMPLE 5

Indirect:antibody capture enzyme linked immunosorbent assay (ELISA)demonstrating affinity of antisera for moiety of Formula (I).

An ELISA was used to screen the antisera for polyclonal antibodies tothe generic hapten/conjugate of the invention using the followingprotocol. Microtitre plates (Costar) were coated with the appropriatehapten conjugate with ovalbumin by diluting that in 0.05M sodiumhydrogen carbonate/sodium carbonate buffer (pH 9.6) to give an optimumprotein concentration of lug/ml previously determined by chequerboardtitration; protein found by Bradford assay. 100μl was added to eachmicrotitre plate well and the plates placed in a moist chamber overnight, washed 3 times with 0.15 phosphate buffered saline pH7.2 with0.05% Tween 20[RTM]: PBST), and blotted dry. Blocking was carried out byadding 250 μl per well of 5% skimmed milk for 1 hour at 25° C.

After two further washings the coated microplates were stored at -20° C.until required, whereon they were washed once with PBST. Antisera werediluted (1/1000 for general screening or doubling dilutions up to1/512,000 for titration assays) in PSBT, applied to microtitre wells intriplicate in 50μl amounts and incubated in a moist chamber at 25° C.for 1 hour (appropriate positive and negative controls wereincluded--PBST, negative rabbit serum and positive serum when it hadbeen produced). Wells were then washed 3 times in PBST and 200 μl of a1:1500 dilution of swine anti-rabbit immunoglobulin conjugated tohorseradish peroxidase (DAKO) was added to each well. Plates wereincubated once more for 1 hour at 25° C. then washed 3 times with PBST.

Tetramethylbenzidine (TMB) enzyme substrate solution was prepared byadding 400μl of 6.0 μl TMB in DMSO to 24.5 ml of 0.1M sodium acetatebuffer pH5.5 with 10μl of 1% H₂ O₂ and 100 μl added to the wells. Themicroplates were incubated for 15 minutes in the dark at 25° C. beforethe reaction was stopped by adding 50 μl of 2.5M H₂ SO₄ and theabsorbance read at 450nm against air. Results: are given below withthose of Example 6.

EXAMPLE 6

Competitive/inhibition assay.

This assay was carried out in the same manner as that of Example 5 withthe following modifications. After plate coating the various dilutionsof antisera were incubated with various concentrations of analytesolution for 1 to 2 hours at 25° C. or overnight at 4° C. Antiserumdilutions tested were 1/1000, 1/2000, 1/5000 and 1/10000 and theconcentrations of the analyte ranged from 200 to 0.OOlpg/ml. Coating ofgeneric hapten-ovalbumin was at 1 μg/ml and later other coatingstrengths were tested ranging from 400 to 12.5 ng/ml. Note: crossreactivity in all cases was checked by carrying out an indirect ELISAwhere plates were coated with the blocking agents or BSA or ovalbumin atlug/ml. Skimmed milk, foetal calf serum (both 5%), caesin (0.3%) or fishskin gelatin (0.5%) were selected as the blocking agent in this regard.Use of competitive assay is preferred for determining cross-reactivity.

Results: Sera collected at 4 and 16 weeks showed reactivity with theconjugate, and chequerboard titrations at 16 weeks (see Table 1 and 2)showed all three rabbit's sera to be strongly reactive. Optimum coatingof wells with the conjugate proved to be achieved with about 1 μg/ml,giving high absorbance with antisera and low absorbance with negatives.It should be noted that cross-reaction with spacer moiety or proteinalone may occur; skimmed milk coated, gelatin blocked plates were shownto obviate this sufficiently for successful ELISA.

Table 3 shows the results of the competitive/inhibition ELISA indicatingthat the coating levels should be less than 1 μg/ml for optimalsensitivity: eg. 400 to 0.012 μg/ml conjugate for 200 to 0.001 μganalyte detection.

                  TABLE 1                                                         ______________________________________                                        Chequerboard absorbance with values for 16 week generic BSA                     conjugate antisera (1/1000) with coating concentrations of 20 to             0.16 μg/ml OP-Ova                                                           Coating Conc.                                                                             Rabbit 19                                                                              Rabbit 20                                                                              Rabbit 21                                                                            Negative                               ______________________________________                                        20.0      2.22     2.30       2.22   0.17                                       10.0 2.15 2.20 2.21 0.16                                                      5.0 2.05 2.21 2.18 0.14                                                       1.25 1.87 2.11 2.06 0.13                                                      0.63 1.51 2.07 1.99 0.14                                                      0.32 1.36 1.94 1.86 0.18                                                      0.16 0.92 1.75 1.41 0.21                                                    ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Absorbance values for 16 week generic BSA conjugate                             antisera screen and dilution when negative (absorbance <0.2) plate           coated at 1 μg/ml with OP-Ova at 1 μg/ml.                                             Absorbance Dilution when negative                                                         Rabbit No. (serum 1/1000) (<0.2)                   ______________________________________                                        19         2.44       1/512000                                                  20 2.56 1/512000                                                              21 2.56 1/512000                                                            ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Competitive/inhibition assay for antisera from                                  rabbits 19, 20, 21 pre-incubated with various concentrations of              unconjugated organophosphate (OP) for 1 hour at 25° C.: 1             μg/ml OP-Ova                                                                   Conc. of OP μg/ml                                                        as preincubated                                                               with antisera Rabbit 19 Rabbit 20 Rabbit 21                                 ______________________________________                                        50.0         1.34        1.19     1.24                                          12.5 1.36 1.31 1.02                                                           3.1 1.50 1.54 1.34                                                            0.4 1.66 1.81 1.45                                                            0.1 1.73 1.95 1.42                                                            0.01 1.83 1.95 1.52                                                         Without OP-  2.30        2.39     --                                            ve serum 0.13 0.13 0.15                                                     ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Competitive/inhibition assay for antisera:preincubated with                     OP for 2 hours at 25° C. Plate coated with 400 μg/ml OP-Ova.        OP conc as preinc'                                                                              Dilution of Rabbit 20 antisera                            with antisera   1/1000    1/2000                                              ______________________________________                                        200.0           0.89      0.69                                                  100.0 0.82 0.67                                                               20.0 0.87 0.67                                                                10.0 0.94 0.74                                                                1.0 1.24 0.87                                                                 0.1 1.48 1.04                                                                 0.01 1.58 1.11                                                                0.001 1.38 1.10                                                             Without OP      2.00      1.69                                                ______________________________________                                    

EXAMPLE 7

Modified competitive inhibition assay.

Assay was carried out in the same manner as Example 5 with the followingmodifications. Antisera was diluted 1:2000 with 1BSA in PBST and 100 μlwas incubated for 2 hours at 25° C. with 100μl of each concentration ofthe unconjugated OP--in this case the generic part-in a microtitrationplate. Concentrations of OP ranged from 200 to 0.0001 μg ml⁻¹.

Cross reactivity of antisera from rabbits 19, 20 and 21 by inhibitionELISA with the various organophosphorus pesticides is shown in Table 5in which concentration (μg ml⁻¹) for 50% inhibition to occur is giventogether with the diluent in which this is achieved--PBST or % methanolin PBST for figures.

                  TABLE 5                                                         ______________________________________                                        +++ strong ++ medium + weak reaction poor-slight                                Inhibitor   Rabbit 19  Rabbit 20  Rabbit 21                                 ______________________________________                                        Hapten + spacer                                                                         +++            +++  (3.0PBST)                                                                             +++                                       Hapten no spacer ++  poor  ++                                                 Spacer - (none) - (none) - (none)                                             Fenitrothion +  +++ (4.8 10%) +                                               Methacrifos +  +++ (8.2PSBT) +                                                Propetamphos +  +++ (36.2 10%) ++                                             Dichlorvos ++  ++ (91.1PSBT) +                                                Dimethanoate - (none) ++ (>150 10%) +                                         Malathion - (none) +  - (none)                                                Chlorfenvinphos - (none) +  +                                                 Etrimfos - (none) +  poor                                                     Chlorpyrifos- poor  poor  poor                                                methyl                                                                        Tetrachlorvinphos - (none) poor  poor                                         Pirimiphos- - (none) poor  - (none)                                           methyl                                                                        Glyphospate - (none) - (none) - (none)                                      ______________________________________                                    

The relative affinities of the various organophosphates for the genericconjugate antibodies as shown by the results from Example 7 are providedbelow as derived by subtracting the amount of inhibition of binding at aconcentration of 0.1 μg/ml organophosphate from that obtained with 100μg/ml organophosphate; results are presented as % of the inhibitionobtained with hapten including spacer as the inhibitor.

                  TABLE 6                                                         ______________________________________                                        Relative inhibition of binding of generic antibodies                            to hapten conjugate as % of hapten + spacer inhibition                            Organophosphate                                                                            Percentage inhibition                                      ______________________________________                                        Hapten-spacer  100.0                                                            Fenitrothion 90.1                                                             Methacrifos 89.1                                                              Propetamphos 75.9                                                             Dichlorvos 60.0                                                               Dimethoate 41.4                                                               Malathion 38.6                                                                Chlorfenvinphos 33.4                                                          Etrimfos 30.8                                                                 Chlorpyrifos-methyl 29.2                                                      Tetrachlorvinphos 27.2                                                        Pirimiphos-methyl 21.1                                                      ______________________________________                                    

What is claimed is:
 1. A hapten compound of formula (II)

    (RO).sub.2 --P(S)--Z--(Y)--B--(D)                          (II)

wherein: R is a lower alkyl, Z is O, S or --NH--, Y is a spacer group, Bis --CO-- or --O--CO-- and D is H or an activator group cabable ofenhancing reaction between the compound and a protein.
 2. A haptencompound as claimed in claim 1 wherein when B is --O--CO-- the activatorgroup is a carbonyl imidazole residue of the type left by activationwith a carbodiimide and when B is CO the activator group is a halogenatom.
 3. A hapten compound as claimed in claim 2 having the formula(III):

    (RO).sub.2 P(S)--O--(Y)--B--(D) (III)

wherein Y is a spacer of four carbon length, with or without sidechains.
 4. A hapten compound as claimed in claim 3 where Y is a--(CH₂)₄-moiety.
 5. A method for synthesis of the haptens of formula (II) and(III), as described in claim 1, wherein a dialkyl halothiophosphate isreacted with the spacer group to produce a hapten.
 6. A hapten compoundof the formula:

    (RO).sub.2 --P(S)--Z--(Y)--B--(D)

wherein R is a lower alkyl, Z is O, S or --NH--, Y is a spacer group offormat --(CH₂)_(n) -- with or without side chains when n provide acarbon chain length of 4 to 6, B is --CO-- or --O--CO--, and D ishydrogen, an activator group enhancing reaction between the compound anda protein or a protein suitable for use in a hapten-protein conjugatefor raising antibodies and antisera.
 7. A hapten compound of formula(II)

    (RO).sub.2 --P--(S)--Z--(Y)--B--(D) (II)

wherein: R, Z, Y, and B are as described in claim 6, and D is hydrogenor an activator group capable of enhancing reaction between the compoundand a protein.
 8. A hapten compound as claimed in claim 7 wherein when Bis --O--CO--, the activator group is a carbonyl imidazole residue of thetype left by activation with a carbodiimide, and when B is CO, theactivator group is a halogen atom.
 9. A hapten compound as claimed inclaim 7 wherein Z is O and Y is a spacer of four carbon length, with orwithout side chains.
 10. A hapten compound as claimed in claim 7 whereinY is a --(CH₂)₄ --.